This invention relates to the production and secretion of proteins which are not ordinarily secreted.
A growing number of recombinant proteins are being developed for therapeutic and diagnostic applications. However, many of these proteins may be difficult or expensive to produce in a functional form and/or in the required quantities using conventional methods. Conventional methods involve inserting the gene responsible for the production of a particular protein into host cells such as bacteria, yeast, or mammalian cells, e.g., COS cells, and then growing the cells in culture media. The cultured cells then synthesize the desired protein. Traditional bacteria or yeast systems may be unable to produce many complex proteins in a functional form. While mammalian cells can reproduce complex proteins, they are generally difficult and expensive to grow, and often produce only mg/L quantities of protein. In addition, non-secreted proteins are relatively difficult to purify from procaryotic or mammalian cells as they are not secreted into the culture medium.
In general, the invention features, a method of making and secreting a protein which is not normally secreted (a non-secreted protein). The method includes expressing the protein from a nucleic acid construct which includes:
(a) a promoter, e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter;
(b) a signal sequence which can direct the secretion of a protein, e.g. a signal sequence from a milk specific protein;
(c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein; and
(d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited.
In preferred embodiments: elements a, b, c (if present), and d are from the same gene; the elements a, b, c (if present), and d are from two or more genes.
In preferred embodiments the secretion is into the milk of a transgenic mammal.
In preferred embodiments: the signal sequence is the xcex2-casein signal sequence; the promoter is the xcex2-casein promoter sequence.
In preferred embodiments the non-secreted protein-coding sequence: is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for glutamic acid decarboxylase or myelin basic protein.
In preferred embodiments, the protein is a mutant protein which lacks a biological activity of the wild type protein.
In another aspect, the invention features, a nucleic acid construct, preferably an isolated nucleic acid construct, which includes:
(a) a promoter, e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter;
(b) a signal sequence which can direct the secretion of a protein, e.g., a signal sequence from a milk specific protein;
(c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein; and
(d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably coupled in the order recited.
In preferred embodiments: elements a, b, c (if present), and d are from the same gene; the elements a, b, c (if present), and d are from two or more genes.
In preferred embodiments the secretion is into the milk of a transgenic mammal.
In preferred embodiments: the signal sequence is the xcex2-casein signal sequence; the promoter is the xcex2-casein promoter sequence.
In preferred embodiments the non-secreted protein-coding sequence: is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for glutamic acid decarboxylase or myelin basic protein.
In preferred embodiments, the protein is inactive, e.g., it is a mutant protein which lacks a biological activity of the wild type protein.
In another aspect, the invention features, a method for providing a non-secreted protein, e.g., a heterologous non-secreted polypeptide, in the milk, of a transgenic mammal. The method includes obtaining milk from a transgenic mammal having introduced into its germline a nucleic acid construct described herein, e.g., a heterologous non-secreted protein-coding sequence operatively linked to a signal and a promoter sequence that result in the preferential expression of the protein-coding sequence in mammary gland epithelial cells, thereby secreting the heterologous non-secreted polypeptide in the milk of the mammal.
In preferred embodiments, the protein is inactive, e.g., it is a mutant protein which lacks a biological activity of the wild type protein.
In preferred embodiments the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats. The preferred transgenic mammal is a goat.
In preferred embodiments, the promoter is selected from the group consisting of the beta lactoglobulin promoter, whey acid protein promoter, xcex2-casein promoter and the lactalbumin promoter. The preferred promoter is the xcex2-casein promoter.
In preferred embodiments, the signal sequence is xcex2-casein signal sequence.
In preferred embodiments, the non-secreted protein-coding sequence: is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for glutamic acid decarboxylase or myelin basic protein; codes for an inactive form of glutamic acid decarboxylase.
In preferred embodiments, the protein is fused to other sequences, e.g., to one or both of: a signal sequence, e.g., the signal sequence of xcex2-casein and/or a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In other preferred embodiments the non-secreted polypeptide is purified from the milk of a transgenic mammal.
In another aspect, the invention features, a method of inducing tolerance in a subject to an antigen. The antigen can be a xenoantigen, an alloantigen or a autoantigen. The antigen can be a protein, e.g., a non-secreted protein.
The method includes:
providing a tolerogen expressed in a transgenic mammal which includes the antigen;
and administering the tolerogen to the subject in an amount sufficient to induce tolerance to said protein antigen.
In preferred embodiments: the tolerogen is administered, preferably orally, to the subject in the milk of a transgenic mammal, e.g., a transgenic dairy mammal, e.g., a goat, sheep, or cow. Other mammals, e.g., pigs, can also be used.
In preferred embodiments, the tolerogen is or includes: a protein, e.g., an inactive protein; a non-secreted protein; a fusion of a non-secreted protein and another peptide sequence, e.g., a protein antigen fused to all or part of a secreted protein.
Preferably, the transgenically produced product, is in inactive form, e.g., it is a mutant which lacks an activity of the wild type protein.
In preferred embodiments, the autoantigen is fused to other sequences, e.g., to one or both of: a signal sequence, e.g., the signal sequence of xcex2-casein and/or a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g. a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In preferred embodiments, the antigen is: an antigen which is characteristic of an autoimmune disorder, e.g., diabetes, lupus, multiple sclerosis, rheumatoid arthritis; GAD; MBP; a transcription factor.
In preferred embodiments the subject is at risk of developing, or has, an autoimmune disorder, e.g., diabetes, lupus, multiple sclerosis, of rheumatoid arthritis.
In yet another aspect, the invention features, a method of treating insulin-dependent diabetes mellittis (IDDM) in a subject. The method includes administering to the subject therapeutically effective amount of a transgenically produced tolerogen which includes an autoantigen, e.g., glutamic acid decarboxylase, or an effective amount of a fusion protein which includes an autoantigen, e.g., glutamic acid decarboxylase.
In preferred embodiments, the subject is orally administered milk from a transgenic mammal which expresses the transgenically produced tolerogen which includes an autoantigen, e.g., glutamic acid decarboxylase, or an effective amount of a fusion protein which includes an autoantigen, e.g., glutamic acid decarboxylase. Preferably, the transgenically produced tolerogen, e.g., glutamic acid decarboxylase is in inactive form, e.g., it is a mutant which lacks an activity of the wild type protein.
In preferred embodiments, the autoantigen is fused to other sequences, e.g., to one or both of: a signal sequence, e.g., the signal sequence of xcex2-casein and/or a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In still another aspect, the invention features treating multiple sclerosis in a subject. The method includes administering to the subject therapeutically effective amount of a transgenically produced tolerogen which includes an autoantigen, e.g., myelin basic protein, or an effective amount of a fusion protein which includes an autoantigen, e.g., myelin basic protein (MBP).
In preferred embodiments, the subject is orally administered milk from a transgenic mammal which expresses the transgenically produced myelin basic protein, or an effective amount of a fusion protein which includes an autoantigen, e.g., myelin basic protein. Preferably, the transgenically produced tolerogen, e.g., MBP is in inactive form, e.g., it is a mutant which lacks an activity of the wild type protein.
In preferred embodiments, the autoantigen is fused to other sequences, e.g., to one or both of: a signal sequence, e.g., the signal sequence of xcex2-casein and/or a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g. a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In another aspect, the invention features, a therapeutic composition which includes a therapeutically effective amount of the transgenieally produced glutamic acid decarboxylase and a pharmaceutically-acceptable carrier or diluent, e.g., the milk of a transgenic mammal. Preferably, the transgenically produced glutamic acid decarboxylase is in inactive form.
In another aspect, the invention features, a therapeutic composition which includes a therapeutically effective amount of the transgenically produced myelin basic protein and a pharmaceutically-acceptable carrier or diluent, e.g., the milk of a transgenic mammal.
In another aspect, the invention features, a fusion protein which includes:
a non-secreted protein, e.g., an inactive non-secreted protein,
a signal sequence which directs the secretion of the protein, e.g., a signal from a secreted protein; and
(optionally) a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein.
In preferred embodiments, the fusion protein includes: the signal sequence and sufficient residues from the amino terminal end of a secreted protein, to allow secretion of the fusion protein in milk, fused to a non-secreted protein. Preferred embodiments include the signal sequence and sufficient residues from the amino terminal end of beta casein, e.g., goat beta casein, fused to a non secreted protein.
In another aspect, the invention features, a composition which includes a tolerogen or other trangenic protein described herein and milk. In preferred embodiments, the milk is the milk of a transgenic mammal which secretes the tolerogen or other protein.
In another aspect, the invention features, a method of inducing tolerance in a transgenic mammal to an antigen. The antigen can be a xenoantigen, an alloantigen or a autoantigen. The antigen can be a protein, e.g., a non-secreted protein, e.g., an inactive protein.
The method includes:
expressing a tolerogen in the milk of the transgenic mammal at a level sufficient to induce tolerance.
In preferred embodiments: the transgenic mammal is a transgenic dairy mammal, e.g., a goat, sheep, or cow. Other mammals, e.g., pigs, can also be used.
In preferred embodiments the tolerogen is or includes: a protein; a protein described herein; a non-secreted protein; a fusion of a non-secreted protein and another peptide sequence, e.g., a protein antigen fused to all or part of a secreted protein.
In preferred embodiments the antigen is: an antigen which is characteristic of an autoimmune disorder, e.g., diabetes, lupus, multiple sclerosis, rheumatoid arthritis; GAD; MBP; a transcription factor.
In another aspect, the invention features, a preparation of milk from a transgenic mammal, which milk includes a protein, e.g., a protein described herein, not normally secreted into the milk of mammals of the species of the transgenic mammal. The transgenic mammal can be a transgenic dairy mammal, e.g., a goat, sheep, or cow. Other mammals, e.g., pigs, can also be used.
In another aspect, the invention features, a transgenically produced non-secreted polypeptide, wherein the polypeptide is secreted.
In preferred embodiments, the transgenically produced non-secreted polypeptide is secreted into the milk of a transgenic mammal.
In preferred embodiments, the transgenically produced non-secreted polypeptide is selected from the group consisting of truncated, nuclear, and cytoplasmic polypeptides. Examples of cytoplasmic polypeptides include, but are not limited to, glutamic acid decarboxylase and myelin basic protein. Preferably, the protein, e.g., glutamic acid decarboxylase, is expressed in an inactive form.
In other preferred embodiments, the transgenic mammal is selected from the group consisting of sheep, mice, pigs, cows and goats. The preferred transgenic mammal is a goat.
In methods herein which induce tolerance by the use of a tolerogen which include a non-secreted protein fragments of the non-secreted protein can be used. It is often desirable that the non-secreted protein lack an activity possessed by the wild type non-secreted protein.
A xe2x80x9csignalxe2x80x9d or xe2x80x9csignal sequence,xe2x80x9d as used herein, refers to an amino terminal sequence which directs the expression of a protein to the exterior of the cell or into a membrane. Preferred signal sequences are those from secreted proteins, more preferably protein which are secreted into the milk of a mammal.
GAD, as used herein, refers to glutamic acid decarboxylase. GAD produced by the GAD65 gene is preferred for use herein.
An xe2x80x9cisolatedxe2x80x9d nucleic acid, as used herein, refers to a nucleic acid molecule which is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5xe2x80x2 and 3xe2x80x2 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an xe2x80x9cisolatedxe2x80x9d nucleic acid, such as a cDNA molecule, can be free of other cellular material.
As used herein, the phrase xe2x80x9cnon-secreted polypeptide,xe2x80x9d refers to a protein which is normally found in the nucleus or the cytoplasm of a cell and which is not normally found as a membrane protein or secreted through the membrane to be released outside the cell. Examples of such proteins include, but are not limited to, enzymes, transcription factors, cell cycle regulatory proteins, oncoproteins, ribosomal proteins, structural proteins, and cellular signal transduction proteins.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
As used herein, a tolerogen, is a molecule which presents an epitope to an organism such that immunological tolerance is induced to the epitope. Tolerogens can be proteins.
As used herein, the term xe2x80x9coperatively linked,xe2x80x9d refers to a DNA segment which is placed into a functional relationship with another DNA segment. For example, DNA for a signal sequence is operatively linked to DNA encoding a polypeptide if it participates in the secretion of the polypeptide; a promoter or enhancer is operatively linked to a coding sequence it is promotes the transcription of the sequence. Generally, DNA sequences that are operatively linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
As used herein, the term xe2x80x9cheterologous polypeptide,xe2x80x9d refers to a protein or peptide coded for by a DNA sequence which is not endogenous to the native genome of the organism in which it is produced, e.g., a mammal in whose milk it is produced. The term also includes a protein or peptide coded for by a DNA sequence which if endogenous to the native genome of the mammal in whose milk it is produced does not lead to the natural production of that protein or peptide in its milk.
The term xe2x80x9csubject,xe2x80x9d as used herein, is intended to include mammals having or being susceptible to an unwanted disease or a condition. Examples of such subjects include humans, dogs, cats, pigs, goats, cows, horses, rats and mice.
The term xe2x80x9ctreating a conditionxe2x80x9d is intended to include preventing, inhibiting, reducing, or delaying the progression of the condition.
The transgenically produced non-secreted polypeptides produced according to the invention find use in a wide variety of therapeutic procedures, such as in preparation of pharmaceutical compositions for administration to patients or in diagnosis of diseases. For example, transgenically produced GAD can be used for diagnosis and treatment of diabetes and transgenically produced MBP can be used in the treatment and diagnosis of multiple sclerosis.
The application of transgenic technology to the commercial production of recombinant proteins in the milk of transgenic animals using milk protein specific signal and promoter sequences offers significant advantages over traditional methods of non-secreted protein production. These advantages include a reduction in the total amount of required capital expenditures, elimination of the need for capital commitment to build facilities early in the product development life cycle, and lower direct production cost per unit for complex proteins. Of key importance is the likelihood that, for certain non-secreted proteins, transgenic production may represent the only technologically and economically feasible method of commercial production.
Myelin basic protein (MBP) is membrane associated protein synthesized by oligodendrocytes and Schwann cells. It is not secreted in its natural environment. MBP is also an autoantigen of the disease multiple sclerosis. Animal model studies and clinical trial data have suggested that administrating MBP orally could establish peripheral immune tolerance and thereby suppress the symptoms of the disease.
Glutamic acid decarboxylase (GAD), another cytoplasmic protein, is an enzyme that catalyzes the biosynthesis of the neurotransmitter, xcex3-aminobutyric acid. The human genome has at least two homologous genes located on chromosomes 2 and 10. The GAD65 and the GAD67 cDNA derived primary amino acid sequences are 65% identical, with the difference between the two isomers in the first 250 amino acids being approximately 75%. GAD65 has been recently identified as a critical xcex2-cell autoantigen in the disease insulin-dependent diabetes mellitus. Experiments in the NOD mouse model of insulin-dependent diabetes mellitus have shown an early appearance of GAD65-reactive antibodies and T cells, and have demonstrated protection of diabetes by early GAD treatment, indicating that GAD65 is a key antigen in the disease process (Kaufman et al. Nature 366:69-72, 1993; Tisch et al. Nature 366:72-5, 1993). The presence of anti-GAD antibodies in the sera of prediabetic individuals can serve as reliable makers for progression to overt diabetes. GAD is, therefore, thought to be a candidate for tolerance therapy.
Current methods of obtaining glutamic acid decarboxylase involve purification from a natural sources such as human or non-human CNS or pancreatic cells (Ortel et al. Brain Res. Bull. 5(2):713-719, 1980; Ortel et al. J. Neurosci. 6:2689-2700, 1981); by preparing synthetic proteins based on the sequence, or by expression in cultured mammalian cells. However, the use of conventionally obtained GAD presents various problems due to the unavailability of large quantities of cells, expense associated with producing synthetic peptides and inability to secrete the recombinant protein in COS cells.
The methods described herein allow the production of high levels of secreted proteins which are not normally secreted, e.g., glutamic acid decarboxylase (GAD), a protein that is a potential therapy for insulin-dependent diabetes mellitus (Type 1 diabetes), in the milk of a transgenic animal.
The expression of GAD, a non-secreted protein, is a significant technical achievement. Proteins that are not normally secreted by cells are extremely difficult to produce as they remain with in the producing cell. In order to generate sufficient quantities of a non-secreted protein, like GAD, the producing cells must be harvested and the protein separated from the cells and other proteins found within those cells.
In a transgenic mammal, the protein is secreted by the mammary gland cells into the milk of the mammal.
The work herein shows the ability of transgenic technology to produce a broad and important class of proteins difficult to express in any other system.
The transgenic production of a non-secreted protein in milk provides a cost-effective method of producing high volumes needed for potential commercialization. In addition, since milk from dairy animals can be ingested by humans, this method is especially appropriate for use in oral tolerization.
Oral tolerance is a mechanism that allows the human body to regulate the immune system so that it can absorb foreign materials as nourishment. Preclinical and clinical observations of oral tolerance show that digesting certain proteins can suppress autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No.: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.